Persulfides have been considered as potential signaling compounds similar to the H2S in "S-persulfidation", a sulfur-mediated redox cycle. The research of this sulfur-mediated species is hindered because of the lack of efficient persulfide donors. In this current study, we have developed one- and two-photon-activated persulfide donors based on an o-nitrobenzyl (ONB) phototrigger, which releases the biologically active persulfide (N-acetyl l-cysteine persulfide, NAC-SSH) in a spatiotemporal manner. Next, we have demonstrated the detection of persulfide release both qualitatively and quantitatively using the well-known "turn on" fluorescence probe, that is, monobromobimane, and the trapping agent, that is, 2,4-dinitrofluorobenzene, respectively. Furthermore, we examined the cytotoxicity of synthesized persulfide donors on HeLa cells and the cytoprotective ability in the highly oxidizing cellular environment.